Nanoliposome-microbubble conjugate including drug for hair loss treatment encapsulated in nanoliposome and composition for alleviating or treating hair loss containing same

ABSTRACT

Provided are a nanoliposome-microbubble conjugate in which a drug for hair loss treatment such as finasteride, minoxidil, dutasteride, etc. is encapsulated in a nanoliposome and a composition for alleviating or treating hair loss containing the same. Since an orally administered agent such as finasteride, currently useful as a drug for hair loss treatment, causes side effects, drug delivery through scalp application is most desirable, but the drug is not delivered to hair follicle cells through scalp application alone. Since a drug for hair loss treatment useful as an external preparation for skin causes various side effects, the concentration thereof that is used needs to be further lowered. The above nanoliposome-microbubble conjugate is capable of increasing the delivery efficiency of a drug for hair loss treatment at a low concentration, and is thus very effective at treating androgenic alopecia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Rule 53(b) Divisional of U.S. application Ser. No.16/646,887 filed Mar. 12, 2020, which is a National Stage ofInternational Application No. PCT/KR2019/004311 filed Apr. 11, 2019,claiming priority based on Korean Patent Application No. 10-2018-0069736filed Jun. 18, 2018, the entire contents of which are incorporatedherein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a nanoliposome-microbubble conjugate inwhich a drug for hair loss treatment is encapsulated in a nanoliposomeand to a composition for alleviating or treating hair loss containingthe same.

More particularly, the present invention relates to ananoliposome-microbubble conjugate, in which a nanoliposome including adrug for hair loss treatment such as finasteride, minoxidil,dutasteride, etc. encapsulated therein is chemically stably conjugatedto a microbubble including a hydrophobic gas therein, and to acomposition for alleviating or treating hair loss containing the same.

BACKGROUND ART

Hair loss generally refers to the loss of thick black hair from thescalp. The causes of hair loss are various, but genetics and the malehormone androgen are considered to be important factors. Of these,androgenic alopecia, which accounts for about 60 to 70% of hair loss,progresses in a manner in which testosterone is converted intodihydrotestosterone (DHT) by 5-alpha reductase (SRDSA) and excessivelyproduced dihydrotestosterone binds to the androgenic receptor (AR) ofdermal papilla cells (DPCs) to thus induce apoptosis, leading to hairloss due to shrinkage of the hair. Since men with high expression of5-alpha reductase, particularly 5-alpha reductase type 2 (SRD5A2), whichis mainly distributed in the dermal papillae and outer root sheath ofhair follicles, or persons having high activity of 5-alpha reductasetype 2 have a quite large amount of dihydrotestosterone compared to mostmen, the possibility of hair loss is increased. Hence, the maintreatment for androgenic alopecia is to lower the amount or activity of5-alpha reductase type 2 in order to prevent the conversion oftestosterone into dihydrotestosterone.

Drugs currently developed as therapeutic agents for hair loss includePropecia, minoxidil, dutasteride, and the like. Propecia, containingfinasteride, functions to directly inhibit 5-alpha reductase type 2, anddutasteride functions to inhibit 5-alpha reductase type 1 and type 2 tothus prevent the conversion of testosterone into dihydrotestosterone,thereby slowing the progression of hair loss. Finasteride is an oraldrug for hair loss treatment, which was originally used to treatprostatic hypertrophy and in which side effects of hair growth wereobserved in patients taking the same, leading to later development as anandrogenic alopecia therapeutic agent. However, side effects such asloss of libido, erectile dysfunction, etc. occur in patients who takesuch a drug, and hair loss resumes when administration of the drug isdiscontinued. In particular, the USFDA recommends that men withinfertility or low sperm counts stop taking the drug (Mysore V et al.,2012).

Meanwhile, two important properties required of intracellular drugdelivery systems are efficiency and cytotoxicity (safety), and carriertechnology of nanoliposomes composed of cholesterols or lipids is widelyused (Zuris J A et al., 2015). However, such nanoliposome technologyalone does not facilitate drug delivery into the dermis, which ispresent below the stratum corneum, which acts as a skin barrier (Nemes Zet al., 1999).

A microbubble, which is an FDA-approved diagnostic ultrasound contrastagent, is provided in the form of a micro-sized bubble filled with ahydrophobic gas. A microbubble technique causes cavitation when themicrobubble is exposed to ultrasound to thus temporarily form pores inthe cell membranes of surrounding cells, whereby the nanoliposome may beeffectively delivered into the cells using sonoporation, through which amaterial penetrates cells through the pores thus formed, unlike othercell delivery methods.

Therefore, the present inventors have prepared a composition in whichfinasteride is encapsulated in a nanoliposome and the nanoliposome isconjugated to a microbubble in order to efficiently deliver thenanoliposome including finasteride encapsulated therein to the dermallayer. Thereby, a carrier having good drug delivery efficiency into thedermis is manufactured and used as a composition for alleviating ortreating hair loss, thus culminating in the present invention.

CITATION LIST Patent Literature

-   (Patent Document 1) Korean Patent No. 10-1683463 (Invention Title:    Microbubble-liposome-melanin nanoparticle complex and contrast agent    comprising the same, Applicant: Seoul National University    Industry-Academic Cooperation Foundation, Registration Date: Dec. 1,    2016)-   (Patent Document 2) Korean Patent No. 10-1082391 (Invention Title:    Nanohybrid composite comprising vitamin C for preventing hair loss    and enhancing hair restoration and composition for enhancing hair    restoration, Applicant: CNPharm Co. Ltd. and Ewha Womans University    Industry-Academic Cooperation Foundation, Registration Date: Nov. 4,    2011)-   (Patent Document 3) Korean Patent No. 10-1054731 (Invention Title:    Nanoparticles containing non-soluble finasteride for    hair-growth-promoting compounds and skin external composition    comprising the same, Applicant: AmorePacific Corp., Registration    Date: Aug. 1, 2011)

Non-Patent Literature

-   (Non-Patent Document 1) Mysore V et al., Finasteride and sexual side    effects, Indian Dermatol. Online J, 2012, 3(1), 62-65.-   (Non-Patent Document 2) Zuris J A et al., Cationic lipid-mediated    delivery of proteins enables efficient protein-based genome editing    in vitro and in vivo, Nat. Biotechnol., 2015, 33(1), 73-80. 1.    Korean Patent No. 10-1452397

DISCLOSURE Technical Problem

An objective of the present invention is to provide ananoliposome-microbubble conjugate in which a drug for hair losstreatment is encapsulated in a nanoliposome and a composition foralleviating or treating hair loss containing the same.

More specifically, an objective of the present invention is to provide ananoliposome-microbubble conjugate, in which a nanoliposome including adrug for hair loss treatment such as finasteride, minoxidil,dutasteride, etc. encapsulated therein is chemically stably conjugatedto a microbubble including a hydrophobic gas therein, and a compositioncontaining the same, which is very effective at alleviating or treatinghair loss while avoiding side effects of oral administration.

Technical Solution

The present invention pertains to a nanoliposome-microbubble conjugate,in which a drug for hair loss treatment is encapsulated in ananoliposome.

The drug for hair loss treatment may include at least one selected fromthe group consisting of finasteride, minoxidil and dutasteride.

Moreover, the drug for hair loss treatment may inhibit the expression oractivity of 5-alpha reductase type 2 to thus prevent the conversion oftestosterone into dihydrotestosterone, or may inhibit the death ofdermal papilla cells.

The nanoliposome may include lecithin, cholesterol and a cationicphospholipid.

The microbubble may include an amphoteric phospholipid, an anionicphospholipid, cholesterol, a cationic phospholipid and adisulfide-group-containing lipid.

The nanoliposome-microbubble conjugate may have a particle size of 1100to 2100 nm.

The present invention may provide a composition for alleviating ortreating hair loss containing the nanoliposome-microbubble conjugate.

In addition, the present invention provides a method of preparing ananoliposome-microbubble conjugate capable of selectively recognizingdermal papilla cells as described below. More preferably, thenanoliposome-microbubble conjugate is prepared by separately preparing ananoliposome and a microbubble and then mixing them.

The nanoliposome may be prepared as follows.

Preferably, the nanoliposome is prepared by: 1) preparing a lipid filmcomposition by mixing lecithin, cholesterol and a cationic phospholipidin chloroform;

2) adding the lipid film composition with a drug solution for hair losstreatment and performing sonication;

3) subjecting the sonicated lipid film composition to freezing andthawing and then to sonication; and

4) centrifuging the lipid film composition sonicated in step 3 andrecovering a nanoliposome that is precipitated.

Also, the microbubble may be prepared as follows.

The microbubble may be prepared by:

A) preparing a lipid film composition by mixing an amphotericphospholipid, cholesterol, an anionic lipid, an amine-group-containinglipid and a disulfide-group-containing lipid in chloroform;

B) adding a glucose solution to step A and performing sonication;

C) subjecting the lipid film composition sonicated in step B to freezingand thawing and then to sonication; and

D) preparing a microbubble by introducing a hydrophobic gas into thelipid film composition sonicated in step C.

A nanoliposome-microbubble conjugate may be formed by mixing themicrobubble thus prepared with the nanoliposome.

Hereinafter, a detailed description will be given of the presentinvention.

The present invention is directed to a nanoliposome-microbubbleconjugate in which a drug for hair loss treatment is encapsulated in ananoliposome and to a composition for alleviating or treating hair losscontaining the same.

The drug for hair loss treatment may be selected from among finasteride,minoxidil, and dutasteride, and any drug for hair loss treatment thathas the same mechanism as these drugs may be used. Moreover, any drugfor hair loss treatment may be used, so long as it is able to inhibitthe expression or activity of 5-alpha reductase type 2 to thus preventthe conversion of testosterone into dihydrotestosterone.

The nanoliposome may include lecithin (α-phosphatidylcholine), acationic phospholipid and cholesterol, thereby forming a membrane thatconstitutes a nanoliposome from the lecithin, cationic phospholipid andcholesterol.

Lecithin is widely distributed in animals/plants and has excellentbiocompatibility, and the stability thereof has been previouslyverified, and thus lecithin is broadly useful in food and medicinecarrier techniques. Furthermore, it may be used as a material thatfacilitates control of the size and change of the shape of thenanoliposome.

The cationic phospholipid may include at least one selected from thegroup consisting of dioleoyl phosphatidylethanolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Preferably useful is1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE).

The nanoliposome of the present invention may be stably dispersed inneutral water, cell broth, blood or the like for several hours or more.

The microbubble of the present invention may include an amphotericphospholipid, an anionic phospholipid, cholesterol, a cationicphospholipid and a disulfide-group-containing lipid, and is prepared byforming a membrane that constitutes the bubble through the hydrophobicgas introduced into the lipid film composition comprising the mixturethereof.

The amphoteric phospholipid may be selected from the group consisting of1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC) and1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC). Preferably,1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) is used.

The anionic phospholipid may include at least one selected from thegroup consisting of dicetyl phosphate (DCP),1,2-dierucoyl-sn-glycero-3-phosphate (DEPA),1,2-dilauroyl-sn-glycero-3-phosphate (DLPA),1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA) and1,2-dioleoyl-sn-glycero-3-phosphate (DOPA). Preferably, dicetylphosphate (DCP) is used.

As the cationic phospholipid, the same cationic phospholipid used forthe preparation of the nanoliposome may be used, and more specifically,the cationic phospholipid may include at least one selected from thegroup consisting of dioleoyl phosphatidylethanolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Preferably,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) is used.

The disulfide-group-containing lipid may be exemplified by1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethyleneglycol)-2000-N-[3-(2-pyridyldithio)propionate, also calledDSPE-PEG-sPDP.

-   -   DSPE-PEG-sPDP 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-

N-poly(ethyleneglycol)-2000-N-[3-(2-pyridyldithio) propionate

The DSPE-PEG-sPDP lipid is a lipid having the chemical structure ofChemical Formula 1 below.

For the preparation of the microbubble, the mixing ratio of theamphoteric phospholipid to the anionic phospholipid to the cholesterolto the cationic phospholipid to the disulfide-group-containing lipid maybe 1-3 mM: 0.1-0.3 mM: 0.5-2 mM: 0.1-0.3 mM: 0.1-0.3 mM. Here, as theamphoteric phospholipid: anionic phospholipid: cholesterol: cationicphospholipid disulfide-group-containing lipid, DPPC: DCP: cholesterol:DPPE: sPDP may be used, and preferably, DPPC: DCP: cholesterol: DPPE:sPDP are mixed at a ratio of 2.0 mM: 0.18 mM: 0.9 mM: 0.17 mM: 0.17 mM.

The DSPE-PEG-sPDP lipid in the microbubble is used as a crosslinkingagent when the microbubble is conjugated to the nanoliposome, thusforming a nanoliposome-microbubble conjugate.

The microbubble may result from bubbling of the mixture of theamphoteric phospholipid, the anionic phospholipid, cholesterol, thecationic phospholipid and the disulfide-group-containing lipid. Theinside of the microbubble may be filled with a hydrophobic gas selectedfrom among SF₆, CO₂, CF₄ and C₃F₈. The hydrophobic gas is preferablySF₆.

The nanoliposome may have a particle size of 100 to 200 nm. If the sizeof the nanoliposome is less than 100 nm, it is difficult to encapsulatethe drug for hair loss treatment into the nanoliposome, and thestability thereof may decrease upon in-vivo injection, which isundesirable. On the other hand, if the size thereof exceeds 200 nm, thecomposition including the nanoliposome may be decreased in stabilityupon in-vivo injection, which is undesirable. Also, the microbubble mayhave a particle size of 1000 to 2000 nm. Accordingly, thenanoliposome-microbubble conjugate may have a particle size of about1100 to 2200 nm.

The present invention may provide a composition for alleviating ortreating hair loss containing the nanoliposome-microbubble conjugatecomposition. The nanoliposome-microbubble conjugate composition iseffective at treating hair loss by inhibiting the male hormone 5areductase and thus lowering the concentration of dihydrotestosterone(DHT).

In the method of preparing the nanoliposome-microbubble conjugatecomposition according to the present invention, the nanoliposome isprepared, the microbubble is prepared, and the nanoliposome and themicrobubble are mixed, thereby yielding a conjugate.

In the preparation of the nanoliposome, lecithin, cholesterol and thecationic phospholipid in step 1 may be mixed at a molar ratio of2:0.01-0.5:0.01-0.5. If the mixing ratio thereof falls out of the aboverange, it is difficult to prepare the lipid that constitutes thenanoliposome.

In step 2, the drug for hair loss treatment may be added at aconcentration of 0.01 to 1000 ng/ml to the lipid film composition. Thesolvent for the drug solution for hair loss treatment may be water oralcohol. Here, the alcohol is preferably ethanol or an aqueous solutionthereof.

The freezing and thawing in step 3 may be repeated 1 to 12 times. Whenthe process of freezing and thawing the lipid film composition isrepeated in this way, a nanoliposome dispersion solution having a moreuniform size may be formed, and the drug encapsulation efficiency of thenanoliposome may increase. If the number of times the process isrepeated exceeds 12, the encapsulation efficiency of the nanoliposomemay decrease. Hence, the above process is preferably performed 12 timesor less.

For conjugation of the nanoliposome to the microbubble, the microbubblemay be stabilized using at least one solution among a glucose solution,glycerol and propylene glycol in step B during the preparation of themicrobubble according to the present invention, and may then be mixedwith the nanoliposome. Preferably, a glucose solution is used. Here, theglucose solution may have a concentration of 1 to 20% (w/v). If theconcentration of glucose exceeds 20% (w/v), the resulting solution maybecome viscous, making it impossible to synthesize the microbubble.

The present invention may provide a pharmaceutical compositioncontaining the nanoliposome-microbubble conjugate. The pharmaceuticalcomposition of the present invention may be formulated into oral dosageforms, such as powder, granule, tablet, capsule, suspension, emulsion,syrup, and aerosol formulations, as well as formulations for externaluse, suppositories, and sterile injectable solutions, in accordance withtypical individual processes. A carrier, an excipient and a diluent thatmay be contained in the pharmaceutical composition may include lactose,dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol,starch, acacia rubber, alginate, gelatin, calcium phosphate, calciumsilicate, cellulose, methylcellulose, microcrystalline cellulose,polyvinylpyrrolidone, water, methylhydroxybenzoate,propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Theformulation may be typically prepared using a diluent or excipient suchas a filler, an extender, a binder, a wetting agent, a disintegrant, asurfactant, and the like. A solid formulation for oral administrationmay include tablets, pills, powders, granules, capsules, and the like,and such a solid formulation may be prepared by mixing the compositionof the present invention with at least one excipient, for examplestarch, calcium carbonate, sucrose, lactose, gelatin, and the like. Inaddition to the simple excipient, lubricants such as magnesium stearate,talc and the like may be used. An oral liquid formulation may includesuspensions, solutions, emulsions, syrups, and the like, and may alsoinclude not only simple diluents, such as water or liquid paraffin, butalso various excipients, for example, wetting agents, sweeteners,fragrances, preservatives, etc. A formulation for parenteraladministration may include sterilized aqueous solutions, non-aqueoussolvents, suspensions, emulsions, freeze-dried preparations andsuppositories. As non-aqueous solvents or suspension agents, propyleneglycol, polyethylene glycol, vegetable oil such as olive oil, injectableesters such as ethyl oleate and the like may be used. As the base of asuppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat,glycerogelatin and the like may be used.

The amount of the pharmaceutical composition according to the presentinvention, when administered, may vary depending on the age, gender andweight of the subject to be treated, the particular disease orpathological condition for treatment, the severity of the disease orpathological condition, the administration route and the judgment of theprescriber. A dose determination based on these factors will be apparentto those skilled in the art, and the dose typically falls in the rangeof 0.01 mg/kg/day to about 2000 mg/kg/day. Preferably, the dose is setto the range of 1 mg/kg/day to 500 mg/kg/day. Administration may becarried out once a day or several times a day. The dose does not in anyway limit the scope of the present invention.

The pharmaceutical composition of the present invention may beadministered to mammals including mice, livestock, humans, and the like,through various routes. All modes of administration, for example,through the skin application, orally, or through intrarectal,intravenous, intramuscular, subcutaneous, intraperitoneal orintracerebroventricular injection, may be considered.

Advantageous Effects

The present invention pertains to a nanoliposome-microbubble conjugatein which a drug for hair loss treatment such as finasteride, minoxidil,dutasteride, etc. is encapsulated in a nanoliposome and to a compositionfor alleviating or treating hair loss containing the same. An orallyadministered agent such as finasteride currently useful as the drug forhair loss treatment causes various side effects when administeredorally, and thus drug delivery through scalp application is regarded asmost desirable, but the drug itself is not delivered well to hairfollicle cells through scalp application alone. In addition, a drug forhair loss treatment useful as an external preparation for skin causesvarious side effects, and therefore the concentration thereof that isused needs to be further lowered.

Accordingly, it is important to increase the drug delivery efficiency offinasteride using a delivery support. The use of thenanoliposome-microbubble conjugate of the present invention is capableof increasing the delivery efficiency of the drug for hair losstreatment at a low concentration, making it possible to effectivelytreat androgenic alopecia.

Korean Patent No. 10-1683463 discloses an intracellular drug deliverytechnique using microbubbles, but it is limited only to technologiesthat can be applied to anticancer drugs. Korean Patent No. 10-1054731discloses nanoparticles including finasteride and an externalpreparation composition for skin containing the same, but the structureof the nanoliposome thereof is different from the present invention,from which it can be confirmed that the present invention is differentfrom the above techniques.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 schematically shows a process by which, when ananoliposome-microbubble conjugate of the present invention is deliveredto dermal papilla cells (DPCs) and sonication is performed, the cellmembrane is perforated and the microbubble collapses, whereby thenanoliposome enters the cells;

FIG. 2 shows a Cryo-EM image of a liposome having a size of 200 nm orless (left) and an optical microscope image of a bubble having a size of1 to 2 μm (right);

FIG. 3 is a photograph showing the intracellular penetration of the drug(including fluorophore) through the nanoliposome-microbubble conjugate(HTP) (right) and a photograph showing the control cells therefor(left), in which the photograph above the cell photograph is anultrasound image of the microbubble (HTP);

FIG. 4 is a graph showing the cytotoxicity of DPCs upon treatment of 3T3cells and DPCs with testosterone (TS) (left), and a graph showing almostno cytotoxicity upon treatment of DPCs with HTP alone or with both HTPand testosterone (TS) (right);

FIG. 5 is photographs showing the results of measurement of hair growtheffects at an interval of one week after inducing hair loss in mice andthen treating the mice five times with the nanoliposome-microbubbleconjugate (HTP) of the present invention (Control: group not treatedwith drug, TS: testosterone, HTS: nanoliposome-microbubble of Example 1,NL: liposome of Comparative Example 1).

MODE FOR INVENTION

A better understanding of the present invention will be given throughthe following examples. However, the present invention is not limited tothe examples described herein, and may be embodied in other forms.Furthermore, the examples are set forth to provide those skilled in theart with an understanding of the spirit of the present invention so thatthe teachings herein are thorough and complete.

Example 1. Production of Nanoliposome-Microbubble Conjugate Example 1-1.Production of Nanoliposome

Lecithin (Sigma Aldrich), cholesterol (Sigma Aldrich) and DPPE (SigmaAldrich) as a cationic phospholipid were mixed at a molar ratio of2:0.1:0.05 in chloroform and then formed into a lipid film using arotary evaporator.

The lipid film was added with finasteride and mixed through sonication.A freezing and thawing cycle using liquid nitrogen was repeated fivetimes, and then sonication (probe mode) was performed, thus preparing auniform nanoliposome composition having a smaller size.

Thereafter, the nanoliposome composition (total amount of lipid: 20.43mg) precipitated through centrifugation was recovered and dispersed in a5% (w/v) glucose aqueous solution. The finasteride concentration in eachof the finasteride solution before the preparation of nanoliposome andthe solution remaining after the preparation of nanoliposome wasmeasured, and thus the drug encapsulation efficiency in the nanoliposomewas found to be 60% or more.

Example 1-2. Production of Microbubble

15.4 mg of DPPC (1,2-dipalmitoyl-sn-glyero-3-phosphocholine, SigmaAldrich) as an amphoteric phospholipid, 3.48 mg of cholesterol (SigmaAldrich), 1 mg of DCP (dicetyl phosphate, Sigma Aldrich) as an anionicphospholipid, 1.2 mg of DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, Sigma Aldrich) as acationic phospholipid, and 5 mg of DSPE-PEG-sPDP(1,2-distearoyl-sn-phosphoethanolamine-N-[PDP (polyethylene glycol)],Avanti polar) as a disulfide-group-containing lipid were mixed in 1 mlof chloroform and then formed into a lipid film for microbubblesynthesis using a rotary evaporator.

Thereafter, 1 ml of a 5% (w/v) glucose aqueous solution was addedthereto and mixed therewith through sonication. A freezing and thawingcycle using liquid nitrogen was repeated three times, and sonication(probe mode) and then filling with an SF₆ gas were conducted, therebypreparing a microbubble composition in a dispersed phase.

Example 1-3. Formation of Nanoliposome-Microbubble

1 ml of the nanoliposome (20.53 mg/ml) prepared in Example 1-1 and 0.5ml of the microbubble (26.08 mg/ml) of Example 1-2 were mixed (at avolume ratio of 2:1), whereby the nanoliposome and the microbubble weredispersed in the glucose aqueous solution.

Thereafter, strong vibration [Mixing frequency: 4500 tr/mn (cpm: m³ permin)] was applied for 15 sec using a machine (Tianjin Iris), thusforming a nanoliposome-microbubble conjugate, which was thenrefrigerated in the state of being dispersed in a 5% glucose aqueoussolution.

The nanoliposome-microbubble conjugate thus obtained is referred to as a‘nanoliposome-microbubble conjugate of Example 1’ or a hair losstherapeutic particle (HTP).

Comparative Example 1. Nanoliposome

A nanoliposome (not conjugated with a microbubble) was prepared in thesame manner as in Example 1-1, and was used as the composition ofComparative Example 1.

Experimental Example 1. Evaluation of Size and Surface Charge ofNanoliposome-Microbubble Conjugate

The nanoliposome and the microbubble produced in Examples 2-1 and 2-2are imaged and shown in FIG. 2. The left image of FIG. 2 shows thenanoliposome and the right image thereof shows the microbubble,indicating that each was prepared at an appropriate size.

The size of the nanoliposome-microbubble conjugate formed by conjugatingthe nanoliposome and the microbubble was measured through dynamic lightscattering (DLS), and the average size thereof was about 1100 nm and thesurface charge was +2.25 mV. The size of the nanoliposome alone ofComparative Example 1 (Example 1-1), not conjugated with themicrobubble, was 98 nm, and the surface charge thereof was +1.75 mV. Inorder to deliver the nanoliposome into dermal papilla cells, a positivesurface charge is preferable. Although the surface charge of themicrobubble conjugate alone was −0.91 mV, the surface charge of thenanoliposome-microbubble conjugate of the present invention was notlowered but had a positive charge of greater magnitude. Therefore, thenanoliposome-microbubble conjugate of the present invention wasconcluded to be suitable for use as a cell delivery composition.

Experimental Example 1. Evaluation of Cell Survival and Penetration

In order to evaluate the entry of the nanoliposome-microbubble conjugateof Example 1 into the dermal papilla cells, DPCs were treated for 2 hrwith the nanoliposome-microbubble conjugate of Example 1 at afinasteride concentration of 100 ng/ml. The confocal fluorescentmicroscope image thereof is shown in FIG. 3. Here, during the productionof the nanoliposome-microbubble conjugate, the nanoliposome lipid wasintroduced with a fluorophore RITC (red) dye and observed.

In FIG. 3, the blue shows a DNA-stained image and the red shows afluorescent image of RITC. No red is observed in the control group, butthe red in the cytoplasm and the blue in the nucleus (DNA) appear mergedin the right image. Therefore, it can be found that the nanoliposomeincluding finasteride encapsulated therein was efficiently introducedinto DPCs due to the treatment with the nanoliposome-microbubbleconjugate of Example 1.

Moreover, the state in which the microbubble was wholly conjugated tothe nanoliposome-microbubble conjugate (HTP) was observed throughultrasound imaging, and it was confirmed that a brown band due to themicrobubble appeared in the HTP solution (the top photograph of FIG. 3).

Experimental Example 3. Evaluation of Cell Viability

Cell viability was evaluated through WST-1 assay (EZ-cytox CellViability Assay Kit). DPCs and 3T3 fibroblasts were cultured at adensity of 1×10⁴/well in a 96-well plate for 24 hr. Here, the culturemedium was replaced with a new medium containing 200 μM testosterone,and after 24 hr, a WST-1 reagent was added thereto. The WST-1 reagentwas added in an amount of 10% of the culture broth, and after 1 hr,absorbance was measured at 460 nm and thus cell survival andproliferation were compared with a control group (non-treated group).Cell survival was measured at an interval of 24 hr for 4 days aftertestosterone treatment.

These results are shown in the left of FIG. 4. The extent of apoptosisover time was greatly increased in DPCs compared to 3T3 cells,indicating that a model of hair loss with an increase in the amount oftestosterone was well established.

After treatment with the nanoliposome-microbubble conjugate (HTP) of thepresent invention before treatment with testosterone in the same manner,the culture medium was replaced with a new medium containing 200 μMtestosterone, the subsequent procedures were conducted as above, and thestate of cells was observed. A comparative group was treated with thenanoliposome-microbubble conjugate (HTP) alone.

As is apparent from the right graph of FIG. 4, the cells grew wellwithout toxicity in the group treated with the nanoliposome-microbubbleconjugate (HTP) alone or the group treated with both thenanoliposome-microbubble conjugate and testosterone (TS+HTP).

These results showed that finasteride was well encapsulated in thenanoliposome-microbubble conjugate of Example 1 of the present inventionand thus the functionality of the above drug was capable of beingoptimally exhibited, and also that the above conjugate was able toeffectively inhibit hair loss due to apoptosis by testosterone in thedermal papilla cells.

Experimental Example 4. Evaluation of Effect of Nanoliposome-MicrobubbleConjugate on Inhibiting Hair Loss in Mouse Model

The hair on the back of each of 6-week-old mice (C57BL/6J) was epilatedusing an animal epilator (Philips) and hair removal cream (Veet), afterwhich testosterone was dissolved in a mixed solution of propylene glycoland ethanol (3:7 (v:v)) and applied every day at a concentration of 30μg/ml, and thus an environment similar to that of human hair loss wasmade.

In the present experiment, the individual test groups were as follows(testosterone was applied to the skin in all cases).

-   -   {circle around (1)} A control group (non-treated group);    -   {circle around (2)} A group treated with testosterone;    -   {circle around (3)} A group treated with testosterone and then        subjected to skin application with the nanoliposome-microbubble        conjugate of Example 1;    -   {circle around (4)} A group treated with testosterone and then        subjected to oral administration with a commercially available        finasteride solution;    -   {circle around (5)} A group treated with testosterone and then        subjected to skin application with a commercially available        finasteride solution;    -   {circle around (6)} A group treated with testosterone and then        subjected to skin application with the nanoliposome of        Comparative Example 1; and    -   {circle around (7)} A group subjected to skin application with        the nanoliposome-microbubble conjugate of Example 1.

A nanoliposome-microbubble conjugate (HTP) configured such that thenanoliposome and the microbubble were conjugated and dispersed at aratio of 2:1 was used. 200 μl thereof, having a total finasteridecontent of 1 μg, was applied on the entire epilated back of the miceusing a plastic spatula, and after 3 min, was exposed to ultrasoundusing a medical sonicator. Here, the nanoliposome-microbubble conjugatewas applied five times at an interval of one day. Under the sameconditions, the total finasteride content was matched in the grouptreated with the commercially available finasteride solution in lieu ofthe solution of Example 1 or Comparative Example 1.

Since the commercially available finasteride solution is an orallyadministered agent, it was administered in a dose 10 times that of theapplication dose in the oral administration group for comparison, andthe administration time and the number of administrations were set thesame as for the skin application.

The results thereof are shown in FIG. 5. In FIG. 5, Control is a groupnot treated with the drug, TS represents testosterone, HTS representsthe nanoliposome-microbubble of Example 1, and NL represents theliposome of Comparative Example 1.

Referring to the results of FIG. 5, the control group ({circle around(1)}) did not undergo any treatment and hair grew naturally over time,and the group ({circle around (2)}) treated with testosterone alone grewhardly any hair.

On the other hand, in the group ({circle around (3)}) treated withtestosterone and then with the composition of Example 1, it wasconfirmed that hair grew well.

In contrast, in the group ({circle around (4)}) treated withtestosterone and then subjected to oral administration with thecommercially available finasteride drug or the group ({circle around(5)}) treated with testosterone and then subjected to skin applicationwith the commercially available finasteride drug, the hair startedgrowing on the seventh week.

In the group ({circle around (6)}) treated with testosterone and thenwith the liposome of Comparative Example 1, not conjugated with themicrobubble, hardly any hair grew, like the comparative group treatedwith testosterone alone. This was confirmed because the drug for hairloss treatment is encapsulated in the liposome and thus the in-vivodelivery ability thereof is lower than that of the commerciallyavailable drug. These results demonstrate that it is essential that theliposome be transported into the biomembrane through the microbubble sothat the drug is delivered.

Although not shown in the photographs of FIG. 5, the hair also grew inthe group treated with the composition (HTP) alone of Example 1 like thecontrol group, and thus, side effects such as special toxicity, etc. didnot appear.

Therefore, the above results suggest that the nanoliposome-microbubbleconjugate of the present invention is very effective at treatingandrogenic alopecia.

What is claimed is:
 1. A method of alleviating or treating hair losscomprising administering a composition containing ananoliposome-microbubble conjugate, in which a drug for hair losstreatment is encapsulated in a nanoliposome.
 2. The method of claim 1,wherein the drug for hair loss treatment comprises at least one selectedfrom the group consisting of finasteride, minoxidil and dutasteride. 3.The method of claim 1, wherein the drug for hair loss treatment inhibitsexpression or activity of 5-alpha reductase type 2 to thus preventconversion of testosterone into dihydrotestosterone, or inhibits deathof dermal papilla cells.
 4. The method of claim 1, wherein thenanoliposome comprises lecithin, cholesterol and a cationicphospholipid.
 5. The method of claim 1, wherein the microbubblecomprises an amphoteric phospholipid, an anionic phospholipid,cholesterol, a cationic phospholipid and a disulfide-group-containinglipid.